1. Field of the Invention
This invention relates to stabilizers for radiopharmaceutical compositions which are added after radiolabeling but prior to administration. Ascorbic acid (and/or a derivative thereof), alone or in combination with other stabilizers, is used to inhibit oxidation loss and autoradiolysis of radiolabeled peptides and proteins.
2. Background Art
A large number of protein-based radiopharmaceuticals are under clinical investigation, and a smaller number have been approved in the United States and other countries. Similarly, peptide-based radiophannaceutieals are also under clinical investigation, with several approved for clinical use. Therapeutic and diagnostic uses of both protein- and peptide-based radiopharmaceuticals continue to be developed. Typical therapeutic and diagnostic applications are described in U.S. Pat. Nos. 5,078,985; 5,102,990; 5,277,893; 5,443,816; and 5,460,785; and in U.S. patent Applications Ser. Nos. 08/087,219; 08/269,929 and 08/651,179, incorporated by reference in their entirety.
Protein- or peptide-based radiopharmaceuticals are primarily based upon use of monoclonal antibodies (or fragments thereof) as a targeting vehicle, but other peptides or proteins can also be used, including albumins and hormones. Both intact antibodies (monoclonal and polyclonal) and fragments, made by any method known to the art, as well as peptide mimics of fragments or antibody binding sites can be radiolabeled and used as imaging, diagnostic or therapeutic agents.
A variety of peptide-based radiopharmaceuticals have been investigated, including those in which the peptide is derived from somatostatin. Radiolabeled peptide analogues of somatostatin used for diagnostic imaging include .sup.123 I-labeled Tyr-3-octreotide and .sup.111 In-diethylene tetraaminepentaacetic acid .sup.99m TPA)-octreotide imaging agents. Research is underway on a variety of .sup.99m Tc-labeled somatostatin analogues, including direct-labeled peptide somatostatin analogues. An .sup.111 In-DTPA-octreotide product is commercially available in the United States and European countries, and is distributed by Mallinckrodt Medical, Inc.
Both protein- and peptide-based radiopharmaceuticals may be radiolabeled by a variety of means. Both peptides and proteins can be directly radioiodinated, through electrophilic substitution at reactive aromatic amino acids. Iodination may also be accomplished via prelabeled reagents, in which the reagent is iodinated and purified, and then linked to the peptide or protein.
The utility of DTPA and EDTA chelates covalently coupled to proteins, polypeptides and peptides is well known in the art. DTPA has been used as a bifunctional chelating agent for radiolabeling a variety of peptides with .sup.111 In, including somatostatin analogues for cancer imaging, .alpha.-melanocyte-simulating hormone for imaging melanoma, chemotactic peptides for infection imaging, laminin fragments for targeting tumor-associated laminin receptors and atrial natriuretic peptides for imaging atrial natriuretic receptors in the kidney.
.sup.99m Tc is a preferred isotope for diagnostic imaging, due to its low cost, ready availability, excellent imaging properties and high specific activities. Two approaches have been described for radiolabeling proteins and peptides with .sup.99m Tc: direct labeling and bifunctional chelates. Direct labeling methods are generally described in U.S. Pat. Nos. 5,078,985; 5,102,990; 5,277,893; 5,443,816; and 5,460,785 referenced above, in which a variety of methods of direct labeling of peptides and proteins through sulfur-, oxygen- and nitrogen-containing amino acid sequences available for binding are disclosed.
A variety of high affinity chelates to bind .sup.99m Tc to specific sites on peptides have been developed. In one approach, the bifunctional reagent is first labeled with .sup.99m Tc, and then conjugated to the peptide. However, multiple species can result, and post-labeling purification is generally required. In another approach, a chelating agent is covalently attached to the peptide prior to radiolabeling. Chelates which have been employed include a variety of N2S2 and N3S ligands, DTPA, 6-hydrazinonicotinate groups, metallothionein and metallothionein fragments.
Isotopes of rhenium, principally .sup.186 Re and .sup.188 Re, have been used to radiolabel proteins and peptides for investigation as therapeutic agents. The chemistry of .sup.186 Re and .sup.188 Re is similar to that of .sup.99m Tc, though not identical, and both direct and chelate labeling approaches have been used in radiolabeling proteins and peptides with rhenium.
Protein and peptide radiopharmaceutical compositions are known to degrade after radiolabeling, primarily by oxidation losses and by autoradiolysis. Some radiopharmaceuticals, such as .sup.99m Tc, and especially .sup.186 Re and .sup.188 Re labeled compounds, are particularly susceptible to oxidation losses if the isotope is not maintained in a suitable oxidation state. Both technetium and rhenium isotopes normally exist in their highest or +7 oxidation state, which is the stable state, until reduced with stannous or other reducing agents. A technetium or rhenium radiolabeled compound can become unstable if the complexed reduced isotope is oxidized to a higher oxidation state, releasing the bound isotope as free or unbound pertechnetate +7 or free perrhenate +7.
The term "autoradiolysis" includes chemical decomposition of a radiolabeled peptide or protein by the action of radiation emitted from the radioisotope coupled to the peptide or protein. Autoradiolysis may be caused by the formation of free radicals in the water or other medium due to the effect of radiation emitted from the radioisotope. Free radicals are molecules or atoms containing a single unpaired electron, which exhibit high chemical reactivity. Autoradiolysis is a significant problem with high energy .beta.-emitting isotopes, such as rhenium isotopes, and with .alpha.-emitting isotopes, but is typically somewhat less of a problem with .gamma.-emitting isotopes, such as .sup.99m Tc.
A variety of methods have been employed to stabilize radiopharmaceuticals in general, including addition of HSA (human serum albumin) to a composition or keeping it frozen between preparation and use. However, these methods are not reliably effective or practical for use with many radiolabeled peptides and proteins. Substances such as ascorbic acid and gentisic acid have also been used to inhibit the oxidation of the radioisotope, and to limit autoradiolysis by acting as "free radical scavengers" which donate reactive hydrogen atoms to the free radical intermediates yielding a non-reactive molecule. Use of gentisic acid and its derivatives to stabilize radiolabeled proteins and peptides is described in U.S. Pat. No. 5,384,113, incorporated herein by reference, and use of ascorbic acid to stabilize some chemical-based radiolabeled compounds, but not protein- or peptide-based radiolabeled compounds, is described in Tofe, A. J. and Francis, M. D., J. Nucl. Med., 17, 820-825 (1976). However, ascorbic acid has been recognized in the art as unsuitable for use as a stabilizing agent with many chemical-based radiolabeled compounds, presumably because it competes for the .sup.99m Tc and forms a .sup.99m Tc-ascorbate complex. Ballinger, J., Der, M., and Bowen, B., Eur. J. Nucl. Med., 6, 154-154 (1981). In fact, because of the stability of Tc-ascorbate complex, ascorbic acid has been labeled with technetium by numerous investigators for use as a potential renal imaging agent. In addition, use of ascorbic acid prior to and during radiolabeling has been described in U.S. Pat. No. 5,011,676, and has been described in Radiopharnaceuticals, G. Subramanian, B. A. Rhodes, J. F. Cooper and V. J. Sodd, eds, Society of Nuclear Medicine, New York, 1975, pp. 37-38, as an agent, used either singly or in combination with Fe(III), in technetium labeling of HSA. Despite the promise shown by a number of newly-developed proteins and peptides for diagnostic and therapeutic applications, susceptibility to oxidation loss, autoradiolysis and other impurities may limit use. Therefore, the development of means for the effective stabilization of radiolabeled compounds, without loss due to the stabilizing agent, is a significant and much-needed advancement in the art.